Method for treating allodynia by peripheral administration of a neurotoxin

ABSTRACT

Methods for treating a non-spasm caused pain by peripheral administration to a patient of a therapeutically effective amount of a neurotoxin, such as a botulinum toxin.

BACKGROUND

The present invention relates to methods for treating pain. Inparticular, the present invention relates to methods for treating painby peripheral administration of a neurotoxin.

Many, if not most ailments of the body cause pain. Generally pain isexperienced when the free nerve endings which constitute the painreceptors in the skin as well as in certain internal tissues aresubjected to mechanical, thermal, chemical or other noxious stimuli. Thepain receptors can transmit signals along afferent neurons into thecentral nervous system and thence to the brain.

The causes of pain can include inflammation, injury, disease, musclespasm and the onset of a neuropathic event or syndrome. Ineffectivelytreated pain can be devastating to the person experiencing it bylimiting function, reducing mobility, complicating sleep, anddramatically interfering with the quality of life.

A muscle spasm can led to stimulation of mechanosensitive pain receptorsthereby causing a sensation of pain. Thus, pain can arise from or be dueto a muscle spasm. Additionally, the spasm can indirectly stimulate thepain receptors by compressing onto blood vessels, causing ischemia inthe tissue, which in turn releases pain inducing substances thatstimulate pain receptors to cause pain sensations. Furthermore, a musclespasm can cause a localized pH reduction which can be perceived as orwhich can engender pain signals. Hence, pain can be a secondary effectof a muscle spasm or muscle hypertonicity.

Inflammatory pain can occur when tissue is damaged, as can result fromsurgery or due to an adverse physical, chemical or thermal event or toinfection by a biologic agent. When a tissue is damaged, a host ofendogenous pain inducing substances, for example bradykinin andhistamine can be released from the injured tissue. The pain inducingsubstances can bind to receptors on the sensory nerve terminals andthereby initiate afferent pain signals.

Additionally, pain inducing substances can be released from nociceptiveafferent terminals, and neuropeptides released from sensory terminalscan accentuate an inflammatory response. Thus, during inflammation therecan be a sprouting of peptidergic peripheral fibers and an increasedcontent of peptide, with many fibers showing a coexistence of substanceP (SP) and calcitonin gene related peptide (CGRP). Substance P caninduce contraction of endothelia cells, which in turn causes plasmaextravasation to allow other substances (bradykinin, ATP, histamine) togain access to the cite of injury and the afferent nerve terminals.Substance P release by the sensory nerve terminal can also degranulatemast cell. This process has been considered to be an important factor inneurogenic inflammation due to the release of inflammatory mediatorssuch as histamine and serotonin and the release of proteolytic enzymeswhich catalyze the production of bradykinin. CGRP apparently does notproduce plasma extravasation but is a powerful vasodilator and also actsynergistically with SP and other inflammatory mediators to enhanceplasma extravasation. All the above listed inflammatory mediators caneither sensitize nociceptors or produce pain.

After activation of the primary sensory afferent neurons the next stepin the transduction of sensory signals can be activation of projectionneurons, which carry the signal, via the spinothalamic tract, to higherparts of the central nervous system such as the thalamic nuclei. Thecell bodies of these neurons (other than those related to the cranialnerves) are located in the dorsal horn of the spinal cord. Here also onecan find the synapses between the primary afferents and the projectionneurons. The dorsal horn is organized into a series of laminae that arestacked, with lamina I being most dorsal followed by lamina II, etc. Thedifferent classes of primary afferents make synapses in differentlaminae. For cutaneous primary afferents, C-fibers make synapses inlaminae I and II, A delta-fibers in laminae I, II, and V, and Abeta-fibers in laminae II, IV, and V. Deeper laminae (V-VII, X) arethought to be involved in the sensory pathways arriving from deepertissues such as muscles and the viscera.

The predominant neurotransmitters at the synapses between primaryafferent neurons and projection neurons are substance P, glutamate, CGRPand neuropeptide Y. The efficiency of transmission of these synapses canbe altered via descending pathways and by local intemeurons in thespinal cord. These modulatory neurons can release a number of mediatorsthat are either inhibitory (e.g. opioid peptides, glycine) or excitatory(e.g. nitric oxide, cholecystokinin), to provide a mechanism forenhancing or reducing awareness of sensations.

Although inflammatory pain is generally reversible and subsides when theinjured tissue has been repaired or the pain inducing stimulus removed,present methods for treating inflammatory pain have many drawbacks anddeficiencies. Thus, the typical oral, parenteral or topicaladministration of an analgesic drug to treat the symptoms of pain or of,for example, an antibiotic to treat inflammatory pain causation factorscan result in widespread systemic distribution of the drug andundesirable side effects. Additionally, current therapy for inflammatorypain suffers from short drug efficacy durations which necessitatefrequent drug re-administration with possible resulting drug resistance,antibody development and/or drug dependence and addiction, all of whichare unsatisfactory. Furthermore, frequent drug administration increasesthe expense of the regimen to the patient and can require the patient toremember to adhere to a dosing schedule.

Examples of treatments for inflammation and muscle pain includenon-steroidal anti-inflammatory drugs (NSAIDS), including aspirin andibuprofen; and opioids, such as morphine.

NSAIDs alleviate pain by inhibiting the production of prostaglandinsreleased by damaged tissues. Prostaglandins have been shown to beperipheral mediators of pain and inflammation, as in arthritic diseases,and a reduction in their concentration provides relief to patients. Ithas been suggested that prostaglandins are involved in the mediation ofpain in the spinal cord and the brain, which may explain the analgesiceffects of NSAIDS in some pain states that do not involve inflammationor peripheral tissue damage. However, prostaglandins are only one ofseveral mediators of pain. As such, NSAIDs have a ceiling of activityabove which increasing doses do not give more pain relief. Furthermore,they have side effects that limit their usefulness. For example, NSAIDscan cause irritation of the gastro-intestinal tract and prolonged usemay lead to the development of extensive ulceration of the gut. This isparticularly true in elderly patients who frequently use NSAIDs fortheir arthritis conditions.

The therapeutic actions of opioids are in the spinal cord. Opioidsinhibit the efficiency of neurotransmission between the primary sensoryafferents (principally C-fibers) and the projection neurons. Theyachieve this by causing a prolonged hyperpolarization of both elementsof these synapses. The use of opioids is effective in alleviating mosttypes of acute pain and chronic malignant pain. There are, however, anumber of chronic malignant pain conditions which are partly orcompletely refractory to opioid analgesia, particularly those whichinvolve nerve compression, e.g. by tumor formation. Unfortunatelyopioids also have unwanted side effects including: (1) depression of therespiratory system, (2) constipation, and (3) psychoactive effectsincluding sedation and euphoria. These side effects occur at dosessimilar to those that produce analgesia and therefore limit the dosesthat can be given to patients. Additionally, opioids such as morphineand heroin are well-known drugs of abuse that lead to physicaldependence, which also involves the development of tolerance. With thedevelopment of tolerance, the dose of a drug required to produce thesame analgesic effect increases with time. This may lead to a conditionin which the doses required to alleviate the pain are life-threateningdue to previously mentioned side-effects.

Although pain arising from inflammation and muscle spasm can beinitiated by mechanical or chemical stimulation of the primary sensoryneuron free terminal, neuropathic pain does not require an initialstimulus to the peripheral, free nerve terminal. Neuropathic pain is apersistent or chronic pain syndrome that can result from damage to thenervous system, the peripheral nerves, the dorsal root ganglion, dorsalroot, or to the central nervous system.

Neuropathic pain syndromes include allodynia, various neuralgias such aspost herpetic neuralgia and trigeminal neuralgia, phantom pain, andcomplex regional pain syndromes, such as reflex sympathetic dystrophyand causalgia. Causalgia is often characterized by spontaneous burningpain combined with hyperalgesia and allodynia.

Tragically there is no existing method for adequately, predictably andspecifically treating established neuropathic pain (Woolf C. et al.,Neuropathic Pain: Aetiology, Symptoms, Mechanisms, and Management,Lancet 1999; 353: 1959-64) as present treatment methods for neuropathicpain consists of merely trying to help the patient cope throughpsychological or occupational therapy, rather than by reducing oreliminating the pain experienced.

For example, current methods to treat neuropathic pain includeadministration of local anesthetic blocks targeted to trigger points,peripheral nerves, plexi, dorsal roots, and to the sympathetic nervoussystem. However, these treatments have only short-lived antinociceptiveeffects. Additionally, longer lasting analgesic treatment methods, suchas blocks by phenol injection or cryotherapy raise a considerable riskof irreversible functional impairment. Furthermore, chronic epidural orintrathecal (collectively “intraspinal”) administration of drugs such asclonidine, steroids, opioids or midazolam have significant side effectsand questionable efficacy.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of a commercially available botulinum toxintype A (purified neurotoxin complex)¹ is a LD₅₀ in mice (i.e. 1 unit).One unit of BOTOX® contains about 50 picograms of botulinum toxin type Acomplex. Interestingly, on a molar basis, botulinum toxin type A isabout 1.8 billion times more lethal than diphtheria, about 600 milliontimes more lethal than sodium cyanide, about 30 million times morelethal than cobra toxin and about 12 million times more lethal thancholera. Singh, Critical Aspects of Bacteral Protein Toxins, pages 63-84(chapter 4) of Natural Toxins II, edited by B. R. Singh et al., PlenumPress, New York (1976) (where the stated LD₅₀ of botulinum toxin type Aof 0.3 ng equals 1 U is corrected for the fact that about 0.05 ng ofBOTOX® equals 1 unit). One unit (U) of botulinum toxin is defined as theLD₅₀ upon intraperitoneal injection into female Swiss Webster miceweighing 18 to 20 grams each.¹ Available from Allergan, Inc., of Irvine, Calif. under the tradenameBOTOX® in 100 unit vials)

Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith type-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain, H chain, and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for tetanustoxin. The carboxyl end segment of the H chain, H_(C), appears to beimportant for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This step is thought to be mediated by the amino end segment ofthe H chain, H_(N), which triggers a conformatioal change of the toxinin response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra-endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxin(or at a minimum the light chain) then translocates through theendosomal membrane into the cytoplasm.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the heavy chain, Hchain, and the light chain, L chain. The entire toxic activity ofbotulinum and tetanus toxins is contained in the L chain of theholotoxin; the L chain is a zinc (Zn++) endopeptidase which selectivelycleaves proteins essential for recognition and docking ofneurotransmitter-containing vesicles with the cytoplasmic surface of theplasma membrane, and fusion of the vesicles with the plasma membrane.Tetanus neurotoxin, botulinum toxin/B/D,/F, and/G cause degradation ofsynaptobrevin (also called vesicle-associated membrane protein VAMP)), asynaptosomal membrane protein. Most of the VAMP present at thecytopiasmic surface of the synaptic vesicle is removed as a result ofany one of these cleavage events. Serotype A and E cleave SNAP-25.Serotype C₁ was originally thought to cleave syntaxin, but was found tocleave syntaxin and SNAP-25. Each toxin specifically cleaves a differentbond (except tetanus and type B which cleave the same bond).

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A has been approved by the U.S. Food andDrug Administration for the treatment of blepharospasm, strabismus andhemifacial spasm. Non-type A botulinum toxin serotypes apparently have alower potency and/or a shorter duration of activity as compared tobotulinum toxin type A. Clinical effects of peripheral intramuscularbotulinum toxin type A are usually seen within one week of injection.The typical duration of symptomatic relief from a single intramuscularinjection of botulinum toxin type A averages about three months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinum typesA and E both cleave the 25 kiloDalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Significantly, it is known that the cytosol of pancreatic islet B cellscontains at least SNAP-25 (Biochem J 1;339 (pt 1): 159-65 (April 1999)),and synaptobrevin (Mov Disord May 1995; 10(3): 376).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 500 kD. complex. Botulinum toxin type D is producedas both 300 kD and 500 kD complexes. Finally, botulinum toxin types Eand F are produced as only approximately 300 kD complexes. The complexes(i.e. molecular weight greater than about 150 kD) are believed tocontain a non-toxin hemaglutinin protein and a non-toxin and non-toxicnonhemaglutinin protein. These two non-toxin proteins (which along withthe botulinum toxin molecule comprise the relevant neurotoxin complex)may act to provide stability against denaturation to the botulinum texinmolecule and protection against digestive acids when toxin is ingested.Additionally, it is possible that the larger (greater than about 150 kDmolecular weight) botulinum toxin complexes may result in a slower rateof diffusion of the botulinum toxin away from a site of intramuscularinjection of a botulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical-preparation will-contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Shantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Shantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1−2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1−2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1−2×10⁷ LD₅₀ U/mg orgreater.

Botulinum toxins and/or botulinum toxin complexes can be obtained fromList Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U.K.; Wako (Osaka,Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals ofSt Louis, Mo.

Pure botulinum toxin is so labile that it is generally not used toprepare a pharmaceutical composition. Furthermore the botulinum toxincomplexes, such a the toxin type A complex are also extremelysusceptible to denaturation due to surface denaturation, heat, andalkaline conditions. Inactivated toxin forms toxoid proteins which maybe immunogenic. The resulting antibodies can render a patient refractoryto toxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin must be stabilized with astabilizing agent. The only successful stabilizing agent for thispurpose has been the animal derived proteins albumin and gelatin. And asindicated, the presence of animal derived proteins in the finalformulation presents potential problems in that certain stable viruses,prions or other infectious or pathogenic compounds carried through fromdonors can contaminate the toxin.

Furthermore, any one of the harsh pH, temperature and concentrationrange conditions required to lyophilize (freeze-dry) or vacuum dry abotulinum toxin containing pharmaceutical composition into a toxinshipping and storage format (ready for use or reconstitution by aphysician) can detoxity some of the toxin. Thus, animal derive or donorpool proteins such as gelatin and serum albumin have been used with somesuccess to stabilize botulinum toxin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown in a medium containingN-Z amine and yeast extract. The botulinum toxin type A complex ispurified from the culture solution by a series of acid precipitations toa crystalline complex consisting of the active high molecular weighttoxin protein and an associated hemagglutinin protein. The crystallinecomplex is re-dissolved in a solution containing saline and albumin andsterile filtered (0.2 microns) prior to vacuum-drying. BOTOX® can bereconstituted with sterile, non-preserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile, vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX® sterile normal saline without apreservative; 0.9% Sodium Chloride Injection is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is believed to be denatured by bubbling or similar violent agitation,the diluent is gently injected into the vial. BOTOX® should beadministered within four hours after reconstitution. During this timeperiod, reconstituted BOTOX® is stored in a refrigerator (2° to 8° C).Reconstituted BOTOX® is clear, colorless and free of particulate matter.The vacuum-dried product is stored in a freezer at or below −5° C.BOTOX® is administered within four hours after the vial is removed fromthe freezer and reconstituted. During these four hours, reconstitutedBOTOX® can be stored in a refrigerator (2° to 8° C.). ReconstitutedBOTOX® is clear, colorless and free of particulate matter.

It has been reported that botulinum toxin type A has been used inclinical settings as follows:

(1) about 75-125 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

-   -   (a) flexor digitorum profundus: 7.5 U to 30 U    -   (b) flexor digitorum sublimus: 7.5 U to 30 U    -   (c) flexor carpi ulnaris: 10 U to 40 U    -   (d) flexor carpi radialis: 15 U to 60 U    -   (e) biceps brachii: 50 U to 200 U. Each of the five indicated        muscles has been injected at the same treatment session, so that        the patient receives from 90 U to 360 U of upper limb flexor        muscle BOTOX® by intramuscular injection at each treatment        session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999, and insome circumstances for as long as 27 months, (The Laryngoscope 109:1344-1346:1999).). However, the usual duration of an intramuscularinjection of Botox® is typically about 3 to 4 months.

As set forth, certain botulinum toxins have been used to treat variousmovement disorders, such as spasmodic muscle conditions with a resultingalleviation of pain. For example, it is known to use a botulinum toxinto treat muscle spasms with resulting relief from both the spasmodicmuscle hyperactivity and from the pain which secondarily arises as aresult of or due to the spasmodic muscle activity. For example, Cheshireet al Pain 1994; 59(1):65-69 reported that patients with myofascial painsyndrome experienced a reduction of pain after injections of botulinumtoxin type A to trigger points. See also WO 94/15629. It is believedthat botulinum toxin A can reduce pain by reducing the sustained musclecontraction that caused or that substantially caused the pain in thefirst place. Thus, the pain which can result from or which can accompanya muscle spasm can be due to the lower, local pH caused by the spasm. Anindirect effect of the flaccid muscle paralysis induced by a botulinumtoxin is to permit the pH to return to a physiological level, therebycausing pain reduction as a secondary effect of the motor endplatecholinergic denervation which can result due to peripheral botulinumtoxin administration.

Botulinum toxin can be used to treat migraine headache pain that isassociated with muscle spasm, vascular disturbances, neuralgia andneuropathy. Binder U.S. Pat. No. 5,714,468, the disclosure of which isincorporated by its entirety herein by reference. Notably, muscle spasmpain, hypertonic muscle pain, myofascial pain and migraine headache paincan all be due, at least in part, to the production and release of oneor more nociceptive substances from the muscles themselves duringperiods of increased muscle tension or contraction.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Astudy of two commercially available botulinum type A preparations(BOTOX® and Dysport®) and preparations of botulinum toxins type B and F(both obtained from Wako Chemicals, Japan) has been carried out todetermine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀ /ED₅₀ . Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg forbotulinum toxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weaknessand duration were dose related for all serotypes. DAS ED₅₀ values(units/kg) were as follows: BOTOX®: 6.7, Dysport®: 24.7, botulinum toxintype B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX® had a longerduration of action than botulinum toxin type B or botulinum toxin typeF. Therapeutic index values were as follows: BOTOX®: 10.5, Dysport: 6.3,botulinum toxin type B: 3.2. Water consumption was greater in miceinjected with botulinum toxin type B than with BOTOX®, althoughbotulinum toxin type B was less effective at weakening muscles. Afterfour months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of4 (where treated with 6.5 ng/kg) rabbits developed antibodies againstbotulinum toxin type B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against botulinum toxin type A. DASresults indicate relative peak potencies of botulinum toxin type A beingequal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX® and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanwas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B. Eur J NeurolNovember 1999;6(Suppl 4):S3-S10.

In addition to having pharmacologic actions at the peripheral location,botulinum oxins may also have inhibitory effects in the central nervoussystem. Work by Weigand et al, Nauny-Schmiedeberg's Arch. Pharmacol.1976; 292, 161-165, and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol.1974; 281, 47-56 showed that botulinum toxin is able to ascend to thespinal area by retrograde transport. As such, a botulinum toxin injectedat a peripheral location, for example intramuscularly, may be retrogradetransported to the spinal cord. However, the authors of the citedarticles were unable to demonstrate that the radioalabelled material wasintact botulinum toxin.

As discussed above, pain associated with muscle disorder, for examplemuscle spasm pain, and headache pain associated with vasculardisturbances, neuralgia and neuropathy may be effectively treated by theuse of botulinum toxin. However, there is a clear deficiency inavailable means for the treatment of an array of other types of pain.Such pain include, for example, pain not associated with muscledisorder, non-headache neuralgia and neuropathy pain, tissueinflammation pain, joint inflammation pain, tissue inflammation pain,cancer pain, post-operational pain, laceration pain, ischemic pain, etc.

Attempts have been made to address these other types of pain, but theirpotential success and possible clinical use is uncertain at this time.For example, Foster et al. in U.S. Pat. No. 5,989,545 (incorporatedherein by reference in its entirety) disclose that a Clostridialneurotoxin, preferably a botulinum toxin, chemically conjugated orrecombinantly fused to a particular targeting moiety can be used totreat pain.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsyrapathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition ofheart rate by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic neurons of the parasympathetic nervoussystem, as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the synapses between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic. The nicotinicreceptors are also present in many membranes of skeletal muscle fibersat the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and parathyroid hormone, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

A neuromuscular junction is formed in skeletal muscle by the proximityof axons to muscle cells. A signal transmitted through the nervoussystem results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscular junction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

What is needed therefore is an effective, long lasting, non-surgicalmethod to treat pain, particularly pain which is not associated with amuscle disorder or headache.

SUMMARY

The present invention meets this need and provides an effective, longlasting, non-surgical method to treat pain, particularly pain which isnot associated with a muscle disorder or headache.

A method within the scope of the present invention for treating pain cancomprise the step of peripheral administration of a neurotoxin to amammal. The pain treated is not associated with a muscle disorder, suchas a muscle spasm, because it is believed that a mechanism by which thepresent invention works is by an antinociceptive effect upon peripheral,sensory afferent pain neurons, as opposed to having an effect upon motorneurons.

The neurotoxin can comprise a neuronal binding moiety which issubstantially native to the neurotoxin. The neurotoxin can be abotulinum toxin, such as one of the botulinum toxin types A, B, C₁, D,E, F or G. Preferably the botulinum toxin is botulinum toxin type A.

The neurotxin can be a modified neurotoxin which has at least one aminoacid deleted, modified or replaced. Additionally, the neurotoxin can bemade at least in part by a recombinant process.

The neurotoxin can be administered in an amount between about 0.01 U/kgand about 35 U/kg and the pain treated can be substantially alleviatedfor between about 1 month and about 27 months, for example for fromabout 1 month to about 6 months.

The peripheral administration of the neurotoxin can be carried out priorto an onset of a nociceptive event or syndrome experienced by a patient.Additionally, the peripheral administration of the neurotoxin can becarried out subsequent to an onset of a nociceptive event experienced bya patient.

A detailed embodiment of a method within the scope of the presentinvention can comprise the step of peripheral administration of abotulinum toxin to a human patient, thereby alleviating pain, whereinthe pain is not associated with a muscle spasm or with a headache.

A further method within the scope of the present invention can comprisethe step of peripheral administration of a neurotoxin to a mammal,wherein the neurotoxin is a polypeptide comprising: (a) a first aminoacid sequence region comprising a wild type neuronal binding moiety,substantially completely derived from a neurotoxin selected from a groupconsisting botulinum toxin types A, B, C₁, D, E, F, G and mixturesthereof; (b) a second amino acid sequence region effective totranslocate the polypeptide or a part thereof across an endosomemembrane, and; (c) a third amino acid sequence region having therapeuticactivity when released into a cytoplasm of a target cell, wherein thepain is not associated with a muscle spasm.

The first amino acid sequence region of the polypeptide can comprise acarboxyl terminal of a heavy chain derived from the neurotoxin and theneurotoxin can be a botulinum toxin, such as botulinum toxin type A.

The second amino acid sequence region of the polypeptide can have anamine terminal of a heavy chain derived from a neurotoxin selected froma group consisting of botulinum toxin types A, B, C₁, D, E, F, G andmixtures thereof. Notably, the second amino acid sequence region of thepolypeptide can include an amine terminal of a toxin heavy chain derivedfrom botulinum toxin type A.

Finally, the third amino acid sequence region of the polypeptide cancomprise a toxin light chain derived from a neurotoxin selected from agroup consisting of Clostridium bertti toxin; butricum toxin; tetanitoxin; botulinum toxin types A, B, C₁, D, E, F, G and mixtures thereof.The third amino acid sequence region of the polypeptide can include atoxin light chain derived from botulinum toxin type A.

The present invention also includes a method for improving patientfunction, the method comprising the step of peripheral administration ofa botulinum toxin to a patient experiencing a non-muscle disorderrelated pain, thereby improving patient function as determined byimprovement in one or more of the factors of reduced pain, reduced timespent in bed, improve hearing, increased ambulation, healthier attitudeand a more varied lifestyle.

Significantly, the neurotoxins within the scope of the present inventioncomprise a native or wild type binding moiety with a specific affinityfor a neuronal cell surface receptor. The neurotoxins within the scopeof the present invention exclude neuronal targeting moieties which arenot native to the neurotoxin because we have found that the presentinvention can be effectively practiced without the necessity of makingany modification or deletions to the native or wild type binding moietyof the neurotoxins used.

Thus, use of a neurotoxin with one or more non-native, targeting moietyartifacts or constructs is excluded from the scope of the presentinvention as unnecessary because, as stated, we have surprisinglydiscovered that peripheral administration of a neurotoxin according tothe present invention provides significant pain alleviation even thoughthe neurotoxin does not comprise a non-native neuronal targeting moiety.Thus we have discovered that a neurotoxin, such as botulinum toxin typeA, can upon peripheral administration provide alleviation of pain eventhough the neurotoxin has not been artificially or manipulativelyaccorded any attachment of a non-native neuronal targeting moiety.

Surprisingly, we have discovered that a neurotoxin, for example aClostridial neurotoxin, having a wild type neuronal binding moiety canbe peripherally administered into a mammal to treat pain. The wild typeneuronal binding moiety is originally part of the neurotoxin. Forexample, botulinum toxin type A, with its original wild type neuronalbinding moiety can be administered peripherally in amounts between about0.01 U/kg to about 35 U/kg to alleviate pain experienced by a mammal,such as a human patient. Preferably, the botulinum toxin used isperipherally administered in an amount between about 0.1 U/kg to about 3U/kg. Significantly, the pain alleviating effect of the presentdisclosed methods can persist for an average of 1-6 months and longer insome circumstances. It has been reported that an effect of a botulinumtoxin can persist for up to 27 months after administration.

In another embodiment, the method of treating pain comprisesadministering to a mammal a neurotoxin, for example a Clostridialneurotoxin, wherein the neurotoxin differs from a naturally occurringneurotoxin by at least one amino acid. The neurotoxin also has a wildtype neuronal binding moiety.

In another embodiment, the methods of treating pain comprisesadministering to a mammal a neurotoxin, for example a Clostridialneurotoxin, wherein the neurotoxin has a wild type neuronal bindingmoiety of another neurotoxin subtype.

The present invention also includes a method for treating apost-operative pain where the pain is a result of the surgical procedurecarried out (i.e. the pain is due, at least in part, to the incisionsmade). The method can comprise the step of peripheral administration ofan effective amount of a botulinum toxin before (i.e. up to 10 daysbefore the surgery), during or immediately after (i.e. by no later thanabout 6-12 hours after the surgery) a surgical procedure, therebyalleviating or significantly alleviating a post-operative pain. Thescope of our invention does not include a method wherein the surgicalprocedure is carried out to treat a muscle spasm.

Our invention also includes a method for treating a visceral pain by anon-systemic, local administration of an effective amount of a botulinumtoxin to thereby alleviate the visceral pain. A visceral pain is a painwhich is perceived by the patient to arise from a site in the viscera,that is in an organ of the digestive, respiratory, urogenital, andendocrine systems, as well in the spleen, heart and/or vessels. Thus,visceral pain includes pain in the pancreas, intestine, stomach andabdominal muscles.

A preferred method within the scope of the present invention fortreating pain comprises the step of peripheral administration of aneurotoxin to a mammal. The pain treated is not substantially due to amuscle spasm because we have surprisingly discovered that a neurotoxinwithin the scope of the present invention can be used to treat painwhich is not secondary to a muscle spasm. Thus, the present invention isapplicable to the treatment of pain which arises irrespective of thepresent or absence of a muscle disorder, such as a muscle spasm.Additionally, the present invention is also applicable to and includeswithin its scope, the treatment of pain which is not secondary to amuscle spasm. Thus, a patient can have a spasmodic or hypertonic muscleand also experience pain which is not secondary, that is does not arisefrom or is not due to, to the muscle spasm. For example, a patient canhave a spasmodic limb muscle and concurrently experience pain in thetruck, such as a back pain. In this example, a method within the scopeof the present invention can treat the back pain by peripheral (i.e.subcutaneous) administration of a neurotoxin to the patient's back.

DEFINITIONS

The following definitions are provided and apply herein:

“Light chain” means the light chain of a clostridial neurotoxin. It canhave a molecular weight of about 50 kDa, and can be referred to as Lchain, L or as the proteolytic domain (amino acid sequence) of aclostridial neurotoxin.

“Heavy chain” means the heavy chain of a clostridial neurotoxin. It canhave a molecular weight of about 100 kDa and can be referred to hereinas H chain or as H.

“H_(N)” means a fragment which can have a molecular weight of about 50kDa, is derived from the H chain of a Clostridial neurotoxin and isapproximately equivalent to the amino terminal segment of the H chain,or the portion corresponding to that fragment in the intact in the Hchain. It is believed to contain the portion of the natural or wild typeclostridial neurotoxin involved in the translocation of the L chainacross an intracellular endosomal membrane.

“H_(C)” means a fragment (about 50 kDa) derived from the H chain of aclostridial neurotoxin which is approximately equivalent to the carboxylterminal segment of the H chain, or the portion corresponding to thatfragment in the intact H chain. It is believed to be immunogenic and tocontain the portion of the natural or wild type Clostridial neurotoxininvolved in high affinity, presynaptic binding to motor neurons.

“Wild type neuronal binding moiety” means that portion of a neurotoxinwhich is native to the neurotoxin and which exhibits a specific bindingaffinity for a receptor on a neuron. Thus, wild type or native neuronalbinding moiety excludes a binding moiety with is not native to theneurotoxin.

“Targeting moiety” means a molecule that has a specific binding affinityfor a cell surface receptor. The targeting moiety is not a Clostridialneurotoxin H_(C), or peptides derived from H_(C) with at least one ofits amino acid deleted, modified or replaced. The targeting moiety is amolecule which is not a Clostridial neurotoxin, for example can be abradykinin.

“Local administration” means administration by a non-systemic route ator in the vicinity of the site of an affliction, disorder or perceivedpain.

“Peripheral administration” means administration by means of anon-systemic route to a peripheral location on a mammal. A peripherallocation generally means, under the skin or into a skeletal muscle.Peripheral administration includes peripheral intramuscular,intraglandular, and subcutaneous administration routes, but excludesintravenous or oral administration and further excludes any directadministration to the central nervous system.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention can become better understood from the following description,claims and the accompanying drawings, where, in FIGS. 1 and 2 below,“injection” means peripheral injection or administration.

FIG. 1 is a dose response graph showing that a method within the scopeof the present invention alleviates induced inflammatory pain under therat formalin model for at least five days. The X axis sets forth time inminutes after commencement of the formalin model in rats. The Y axissets forth time spent lifting and licking the formalin injected paw uponuse of control (saline, n=7) and BOTOX® (botulinum toxin type A purifiedneurotoxin complex) injections at concentrations of 7 U/kg (n=8), 15U/kg (n=5) and 30 U/kg (n=4). The BOTOX® was injected 5 days beforecommencement of the formalin challenge.

FIG. 2 is a dose response graph showing that a method within the scopeof the present invention alleviates induced inflammatory pain under therat formalin model for at least twelve days. The X axis sets forth timein minutes after commencement of the formalin model in rats. The Y axissets forth time spent lifting and licking the formalin injected paw uponuse of control (saline, n=3) and BOTOX® (botulinum toxin type A purifiedneurotoxin complex) injections at concentrations of 3.5 U/kg (n=7) and 7U/kg (n=8). The BOTOX® was injected 12 days before commencement of theformalin challenge.

Description

The present invention is based on the discovery that peripheraladministration of a neurotoxin can provide effective treatment ofchronic pain. Notably, the neurotoxin has a wild type or native neuronalbinding moiety. The pain treated is not due to a muscle spasm, nor isthe pain headache pain. Chronic pain is treated because of the long termantinociceptive effect of the neurotoxins used. The neuronal bindingmoiety component of the neurotoxin is a neuronal binding moiety which isnative to the selected neurotoxin because we have discovered that thepresent invention can be practiced without replacement of the wild typeneuronal binding moiety with a non-native or non wild type targetingmoiety. Treatment of headache pain is not within the scope of thepresent invention because the preferred sites of peripheraladministration of a neurotoxin according to the present inventionexclude the head and neck.

Prior to our discovery a neurotoxin, such as a botulinum toxin, has beenused to treat pain associated with various muscle disorders. Thus, it isknown that a muscle disorder, such as a spasmodic muscle, can cause painand that by treating the spasm the pain can also be alleviated. Fosteret al. discloses that the neurotoxin be linked to a targeting moiety foruse in the treatment of pain, that is that the wild type binding moietyof a Clostridial neurotoxin be removed completely, and replaced by atargeting moiety.

Surprising we have discovered that a neurotoxin which has not beenconjugated, attached, adhered to or fused with a neuronal targetingmoiety can be peripherally administered according to the methods of thepresent invention to treat pain. Preferably, the pain treated is notdue, that is the pain does not directly arise from as a secondary resultof, a muscle spasm. Our invention can be used to treat pain whichresults from a wide variety of neuropathic, inflammatory, cancerous andtrauma conditions.

Prior to our invention is was not known that a neurotoxin, such as abotulinum toxin, could be used to effectively treat pain, where the painis not due to a muscle spasm or hypertonic muscle condition. Thephysiological mechanism by which peripheral administration of aneurotoxin can result in long term alleviation of pain is unclear. Wenote that whereas the pain due to a muscle spasm or hypertonic musclecondition can produce a reduced, local pH, our invention does not restupon and does not require elevation of a local, low pH level.Additionally, whereas a muscle spasm or hypertonic muscle condition canbe alleviated by an anticholinergic effect of a neurotoxin, such as abotulinum toxin, upon motor neurons, our invention is not predicatedupon an effect upon motor neurons. Without wishing to be bound bytheory, we hypothesize, that one effect of peripheral administration ofa neurotoxin, such as a botulinum toxin, according to the presentinvention can be an antinociceptive effect upon a peripheral, sensoryafferent neuron. Significantly, in our invention pain alleviation is aprimary, as opposed to being a secondary, effect upon peripheraladministration of a neurotoxin, such as a botulinum toxin.

Thus, the present invention is based, at least in part, upon thediscovery that a neurotoxin having a wild type neuronal binding moietymay be peripherally administered to a mammal to alleviate pain. Theneurotoxin according to this invention is not coupled to a non-nativetargeting moiety. A wild type binding moiety according to the presentinvention can be a naturally existing H_(C) segment of a Clostridialneurotoxin or an amino acid sequence substantially completely derivedfrom the H_(C) segment of the Clostridial neurotoxin.

As used hereinafter, an amino acid sequence, for example a wild typebinding moiety, “derived from” another amino acid sequence, for examplethe H_(C) segment, means that the resultant amino acid sequence isduplicated exactly like the amino acid sequence from which it isderived; or the resultant amino acid sequence has at least one aminoacid deleted, modified or replaced as compared to the amino acidsequence from which it is derived.

According to one broad aspect of the invention, there are providedmethods for treatment of pain which comprise administering to a mammaleffective doses of a neurotoxin, for example a Clostridial neurotoxin,having a wild type neuronal binding moiety. In one embodiment, themethods include administering to a mammal a neurotoxin having a wildtype neuronal binding moiety which is originally already a part of theneurotoxin. For example, such neurotoxin may be selected from a groupconsisting of beratti toxin and butyricum toxin, each of which alreadyhas a neuronal binding moiety. The neurotoxin may also be a tetanitoxin, which also has a wild type neuronal binding moiety. Preferably,the neurotoxin administered to the mammal is selected from a groupconsisting of botulinum toxin types A, B, C₁, D, E, F, or G, each ofwhich has its own original wild type neuronal binding moiety. Morepreferably, the methods include the administration of botulinum type Awith its original wild type neuronal binding moiety. The methods alsoinclude the administration of a mixture of two or more of the aboveneurotoxins to a mammal to treat pain.

In another embodiment, the methods comprise the administration of aneurotoxin, for example a Clostridial neurotoxin, to a mammal whereinthe neurotoxin differs from a naturally occurring neurotoxin by at leastone amino acid. For example, variants of botulinum toxin type A asdisclosed in Biochemistry 1995, 34, pages 15175-15181 and Eur. J.Biochem, 1989, 185, pages 197-203 (incorporated herein by reference inits entirety) may be administered to a mammal to treat non-spasm relatedpain. These variants also have wild type neuronal binding moieties.

In another embodiment, methods are provided for an administration of aneurotoxin to a mammal to treat non-spasm caused pain, wherein theneurotoxin has a wild type neuronal binding moiety of anotherneurotoxin. For example, the method includes the step of administeringto a mammal botulinum toxin type A having a wild type neuronal bindingmoiety of botulinum toxin type B. All other such combinations areincluded within the scope of the present invention.

In another broad embodiment, methods of the present invention to treatnon-spasm related pain include local, peripheral administration of theneurotoxin to an actual or a perceived pain location on the mammal. Inone embodiment, the neurotoxin is administered subcutaneously at or nearthe location of the perceived pain, for example at or near a chronicallypainful joint. In another embodiment, the neurotoxin is administeredintramuscularly at or near the location of pain, for example at or neara neoplasm on the mammal. In another embodiment, the neurotoxin isinjected directly into a joint of a mammal, for treating or alleviatingpain causing arthritic conditions. Also, frequent, repeated injectionsor infusion of the neurotoxin to a peripheral pain location is withinthe scope of the present invention. However, given the long lastingtherapeutic effects of the present invention, frequent injections orinfusion of the neurotoxin may not be necessary. For example, practiceof the present invention can provide an analgesic effect, per injection,for 2 months or longer, for example 7 months, in humans.

Without wishing to limit the invention to any mechanism or theory ofoperation, it is believed that when the neurotoxin is administeredlocally to a peripheral location, it inhibits the release ofneuro-substances, for example substance P, from the peripheral primarysensory terminal. As discussed above, a release of substance P by theperipheral primary sensory terminal may cause or at least amplify paintransmission process. Therefore, inhibition of its release at theperipheral primary sensory terminal will dampen the pain transmissionprocess.

In addition to having pharmacologic actions at a peripheral location ofadministration, a method within the-scope of the present invention mayalso have an antinociceptive effect due to retrograde transport to theneurotoxin from the site of peripheral (i.e. subcutaneous) injection tothe central nervous system. We have determined that botulinum type A canbe retrograde transported from the peripheral site of administrationback to the dorsal horn of the spinal cord. Presumably the retrogradetransport is via the primary afferent. This finding is consistent withthe work by Weigand et al, Nauny-Schmiedeberg's Arch. Pharmacol. 1976;292, 161-165, and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974;281, 47-56, which showed that botulinum toxin is able to ascend to thespinal area by retrograde transport. Thus, it was reported thatbotulinum toxin type A injected intramuscularly may be retrogradetransported from the peripheral primary sensory terminal to the centralprimary sensory terminal.

Our discovery differs significantly from the discussion in the articlescited in the paragraph above. We have discovered that, in the rat, afterperipheral, subcutaneous administration botulinum toxin was foundlocalized in the animal's dorsal horn, that is at the location where theC fibers synapse. A subcutaneous injection is an injection at a locationwhere many bipolar nociceptive nerve fibers are located. These sensoryfibers run from the periphery to the dorsal horn of the spinal cord.Contrarily, in one or more of the articles cited in the paragraph aboveafter intramuscular toxin injection was carried out some radioalabelledbotulinum toxin was found localized in the ventral roots. The ventralroot of the spinal cord is where monopolar efferent (traffic out) motorneurons are located. Thus, the art leads to an expectation thatperipheral muscle spasticity can be expected as a result of retrogradetransport of a botulinum toxin from the periphery to a spinal cordlocation.

Thus, it had been believed by those skilled in the art that theappearance of a neurotoxin, such as a botulinum toxin in the spinal cordof a mammal would: (1) induce significant spasticity in the recipient,and; (2) promote detrimental effects upon spinal cord and brainfunctions. Thus, with regard to cited deleterious effect (1): it wasreported, as examples, in Wiliamson et al., in Clostridial Neurotoxinsand Substrate Proteolysis in Intact Neurons, J. of Biological Chemistry271:13; 7694-7699 (1996) that both tetanus toxin and botulinum toxintype A inhibit the evoked release of the neurotransmitters glycine andglutamate from fetal mice spinal cord cell cultures, while it wasreported by Hagenah et al., in Effects of Type A Botulinum Toxin on theCholinergic Transmission at Spinal Renshaw Cells and on the InhibitoryAction at la Inhibitory Intemeurones, Naunyn-Schmiedeberg's Arch.Pharmacol. 299, 267-272 (1977), that direct intraspinal injection ofbotulinum toxin type A in experimentally prepared, anaesthetized catsinhibits CNS Renshaw cell activity. Inhibition of central glycine andglutamate neurotransmitter release as well as the downregulation ofRenshaw cell activity presumably can both result in vivo in thepromotion of significant motorneuron hyperactivity with ensuingperipheral muscle spasticity.

With regard to deleterious effect (2): it is believed that central(spinal cord) presence of a tetanus neurotoxin exerts, by retrogrademovement of the tetanus toxin along CNS neurons, significant negativeeffects upon spinal cord and brain functions, thereby contraindicatingany desire to have a related neurotoxin, such as a botulinum toxinappear (as by retrograde transport) in the spinal cord. Notably,botulinum toxin and tetanus toxin are both made by Clostridial bacteria,although by different species of Clostridium. Significantly, someresearchers have reported that botulinum toxin shares, at least to someextent, the noted neural ascent characteristic of tetanus toxin. Seee.g. Habermann E., ¹²⁵ I-Labeled Neurotoxin from Clostridium BotulinumA: Preparation, Binding to Synaptosomes and Ascent in the Spinal Cord,Naunyn-Schmiedeberg's Arch. Pharmacol. 281, 47-56 (1974).

Our invention surprisingly encounters neither of the deleterious effects(1) or (2), and the disclosed peripheral (subcutaneous) administrationmethods of the present invention can be practiced to provide effectiveand long lasting relief from pain which is not due to a muscle spasm andto provide a general improvement in the quality of life experienced bythe treated patient. The pain experienced by the patient can be due, forexample, to injury, surgery infection, accident or disease (includingcancer and diabetes), including neuropathic diseases and disorders,where the pain is not primarily due to a muscle spasm or hypertonicmuscle condition.

Once in the central primary sensory terminal located in the dorsal hornof the spinal chord, the neurotoxin may further inhibit the release ofthe neurotransmitter responsible for the transmission of pain signals,for example substance P. This inhibition prevents the activation of theprojection neurons in the spinothalamic tract and thereby alleviatingpain. Therefore, the peripheral administration of the neurotoxin, due toits now discovered central antinociceptive effect, serve as analternative method to central (i.e. intraspinal) administration of ananalgesic, thereby eliminating the complications associated with centraladministration of an analgesic drug.

Furthermore, it has been shown by Habermann Experientia 1988; 44:224-226that botulinum toxin can inhibit the release of noradrenalin and GABAfrom brain homogenates. This finding suggests that botulinum toxin canenter into the adrenergic sympathetic nerve terminals and GABA nerveterminals. As such, botulinum toxin can be administered to thesympathetic system to provide long term block and alleviate pain, forexample neuropathic pain. The administration a neurotoxin, preferablybotulinum toxin type A, provides a benefit of long term block withoutthe risk of permanent functional impairment, which is not possible withpharmaceutics currently in use.

The amount of the neurotoxin administered can vary widely according tothe particular disorder being treated, its severity and other variouspatient variables including size, weight, age, and responsiveness totherapy. For example, the extent of the area of peripheral pain isbelieved to be proportional to the volume of neurotoxin injected, whilethe quantity of the analgesia is, for most dose ranges, believed to beproportional to the concentration of neurotoxin injected. Furthermore,the particular location for neurotoxin administration can depend uponthe location of the pain to be treated.

Generally, the dose of neurotoxin to be administered will vary with theage, presenting condition and weight of the mammal to be treated. Thepotency of the neurotoxin will also be considered.

In one embodiment according to this invention, the therapeuticallyeffective doses of a neurotoxin, for example botulinum toxin type A, ata peripheral location can be in amounts between about 0.01 U/kg andabout 35 U/kg. A preferred range for administration of a neurotoxinhaving a wild type neuronal binding moiety, such as the botulinum toxintype A, so as to achieve an antinociceptive effect in the patienttreated is from about 0.01 U/kg to about 35 U/kg. A more preferred rangefor peripheral administration of a neurotoxin, such as botulinum toxintype A, so as to achieve an antinociceptive effect in the patienttreated is from about 1 U/kg to about 15 U/kg. Less than about 0.1 U/kgcan result in the desired therapeutic effect being of less than theoptimal or longest possible duration, while more than about 2 U/kg canstill result in some symptoms of muscle flaccidity. A most preferredrange for peripheral administration of a neurotoxin, such as thebotulinum toxin type A, so as to achieve an antinociceptive effect inthe patient treated is from about 0.1 U/kg to about 1 U/kg.

Although examples of routes of administration and dosages are provided,the appropriate route of administration and dosage are generallydetermined on a case by case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see forexample, Harrison's Principles of Internal Medicine (1998), edited byAnthony Fauci et al., 14^(th) edition, published by McGraw Hill). Forexample, the route and dosage for administration of a neurotoxinaccording to the present disclosed invention can be selected based uponcriteria such as the solubility characteristics of the neurotoxin chosenas well as the intensity of pain perceived.

In another broad embodiment of the invention, there are provided methodsfor treating non-spasm related pain which comprises administeringeffective doses of a neurotoxin, wherein the neurotoxin is a singlepolypeptide as opposed to a di-polypeptide as described above.

In one embodiment, the neurotoxin is a single polypeptide having threeamino acid sequence regions. The first amino acid sequence regionincludes a neuronal binding moiety which is substantially completelyderived from a neurotoxin selected from a group consisting of berattitoxin; butyricum toxin; tetani toxin; botulinum toxin types A, B, C₁, D,E, F, and G. Preferably, the first amino acid sequence region is derivedfrom the carboxyl terminal of a toxin heavy chain, H_(C). Morepreferably, the first amino acid sequence region is derived from theH_(C) of botulinum toxin type A.

The second amino acid sequence region is effective to translocate thepolypeptide or a part thereof across an endosome membrane into thecytoplasm of a neuron. In one embodiment, the second amino acid sequenceregion of the polypeptide comprises an amine terminal of a heavy chain,H_(N), derived from a neurotoxin selected from a group consisting ofberatti toxin; butyricum toxin; tetani toxin; botulinum toxin types A,B, C₁, D, E, F, and G. Preferably, the second amino acid sequence regionof the polypeptide comprises an amine terminal of a toxin heavy chain,H_(N), derived botulinum toxin type A.

The third amino acid sequence region has therapeutic activity when it isreleased into the cytoplasm of a target cell or neuron. In oneembodiment, the third amino acid sequence region of the polypeptidecomprises a toxin light chain, L, derived from a neurotoxin selectedfrom a group consisting of beratti toxin; butyricum toxin; tetani toxin;botulinum toxin types A, B, C₁, E, F, and G. Preferably, the third aminoacid sequence region of the polypeptide comprises a toxin light chain,L, derived from botulinum toxin type A.

In one embodiment, the polypeptide comprises a first amino acid sequenceregion derived from the H_(C) of the tetani toxin, a second amino acidsequence region derived from the H_(N) of botulinum toxin type B, and athird amino acid sequence region derived from the L chain of botulinumtype A. In a preferred embodiment, the polypeptide comprises a firstamino acid sequence region derived from the H_(C) of the botulinum toxintype B, a second amino acid sequence region derived from the H_(N) Ofbotulinum toxin type A, and a third amino acid sequence region derivedfrom the L chain of botulinum type A. All other such combinations areincluded within the scope of the present invention.

In another embodiment, the polypeptide comprises a first amino acidsequence region derived from the H_(C) of the botulinum toxin type A,wherein the amino acid sequence has at least one amino acid deleted,modified or replace; a second amino acid sequence region derived fromthe H_(N) of botulinum toxin type A, and a third amino acid sequenceregion derived from the L chain of botulinum type A. All other suchcombinations are Included within the scope of the present invention.

As indicated above, these polypeptides are single chains and may not beas potent as desired. To increase their potency, the third amino acidsequence region may be cleaved off by a proteolytic enzyme, for examplea trypsin. The independent third amino acid sequence region may bereattached to the original polypeptide by a disulfide bridge. In oneembodiment, the third amino acid sequence region is reattached theoriginal polypeptide at the first amino acid sequence region. In apreferred embodiment, the third amino acid sequence region is reattachedto the second amino acid sequence region.

If an unmodified neurotoxin is to be used to treat non-spasm relatedpain as described herein, the neurotoxin may be obtained by culturing anappropriate bacterial species. For example botulinum toxin type A can beobtained by establishing and growing cultures of Clostridium botulinumin a fermenter and then harvesting and purifying the fermented mixturein accordance with known procedures. All the botulinum toxin serotypesare initially synthesized as inactive single chain proteins which mustbe cleaved or nicked by proteases to become neuroactive. The bacterialstrains that make botulinum toxin serotypes A and G possess endogenousproteases and serotypes A and G can therefore be recovered frombacterial cultures in predominantly their active form. In contrast,botulinum toxin serotypes C₁, D and E are synthesized by nonproteolyticstrains and are therefore typically unactivated when recovered fromculture. Serotypes B and F are produced by both proteolytic andnonproteolytic strains and therefore can be recovered in either theactive or inactive form. However, even the proteolytic strains thatproduce, for example, the botulinum toxin type B serotype only cleave aportion of the toxin produced. The exact proportion of nicked tounnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the botulinum toxin type B toxin is likelyto be inactive, possibly accounting for the known significantly lowerpotency of botulinum toxin type B as compared to botulinum toxin type A.The presence of inactive botulinum toxin molecules in a clinicalpreparation will contribute to the overall protein load of thepreparation, which has been linked to increased antigenicity, withoutcontributing to its clinical efficacy. Additionally, it is known thatbotulinum toxin type B has, upon intramuscular injection, a shorterduration of activity and is also less potent than botulinum toxin type Aat the same dose level.

If a modified neurotoxin is to be used according to this invention totreat non-spasm related pain, recombinant techniques can be used toproduce the desired neurotoxins. The technique includes steps ofobtaining genetic materials from natural sources, or synthetic sources,which have codes for a neuronal binding moiety, an amino acid sequenceeffective to translocate the neurotoxin or a part thereof, and an aminoacid sequence having therapeutic activity when released into a cytoplasmof a target cell, preferably a neuron. In a preferred embodiment, thegenetic materials have codes for the H_(C), H_(N) and L chain of theClostridial neurotoxins, modified clostridial neurotoxins and fragmentsthereof. The genetic constructs are incorporated into host cells foramplification by first fusing the genetic constructs with a cloningvectors, such as phages or plasmids. Then the cloning vectors areinserted into hosts, preferably E. coli's. Following the expressions ofthe recombinant genes in host cells, the resultant proteins can beisolated using conventional techniques.

Although recombinant techniques are provided for the production modifiedneurotoxins, recombinant techniques may also be employed to producenon-modified neurotoxins, for example botulinum toxin A as it existsnaturally, since the genetic sequence of botulinum toxin type A isknown.

There are many advantages to producing these neurotoxins recombinantly.For example, production of leurotoxin from anaerobic Clostridiumcultures is a cumbersome and time-consuming process including amulti-step purification protocol involving several protein precipitationsteps and either prolonged and repeated crystallization of the toxin orseveral stages of column chromatography. Significantly, the hightoxicity of the product dictates that the procedure must be performedunder strict containment (BL-3). During the fermentation process, thefolded single-chain neurotoxins are activated by endogenous clostridialproteases through a process termed nicking. This involves the removal ofapproximately 10 amino acid residues from the single-chain to create thedichain form in which the two chains remain covalently linked throughthe intrachain disulfide bond.

The nicked neurotoxin is much more active than the unnicked form. Theamount and precise location of nicking varies with the serotypes of thebacteria producing the toxin. The differences in single-chain neurotoxinactivation and, hence, the yield of nicked toxin, are due to variationsin the type and amounts of proteolytic activity produced by a givenstrain. For example, greater than 99% of Clostridial botulinum type Asingle-chain neurotoxin is activated by the Hall A Clostridial botulinumstrain, whereas type B and E strains produce toxins with lower amountsof activation (0 to 75% depending upon the fermentation time). Thus, thehigh toxicity of the mature neurotoxin plays a major part in thecommercial manufacture of neurotoxins as therapeutic agents.

The degree of activation of engineered clostridial toxins is, therefore,an important consideration for manufacture of these materials. It wouldbe a major advantage if neurotoxins such as botulinum toxin and tetanustoxin could be expressed, recombinantly, in high yield inrapidly-growing bacteria (such as heterologous E. coli cells) asrelatively non-toxic single-chains (or single chains having reducedtoxic activity) which are safe, easy to isolate and simple to convert tothe fully-active form.

With safety being a prime concern, previous work has concentrated on theexpression in E. coli and purification of individual H and L chains oftetanus and botulinum toxins; these isolated chains are, by themselves,non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994); Zhou et al.,Biochemistry 34:15175-15181 (1995), hereby incorporated by referenceherein. Following the separate production of these peptide chains andunder strictly controlled conditions the H and L chains can be combinedby oxidative disulphide linkage to form the neuroparalytic di-chains.

It is known that post operative pain resulting from (i.e. secondary to)a muscle spasm can be alleviated by pre-operative injection of botulinumtoxin type A. Developmental Medicine & Child Neurology 42;116-121:2000.Contrarily, our invention encompasses a method for treatingpostoperative pain by pre or peri-operative, peripheral administrationof a botulinum toxin where the pain is not due to a spasmodic muscle.

Thus, a patient can either during surgery or up to about ten days priorto surgery (where the surgery is unrelated to correction of or treatmentof a spasmodic muscle condition) be locally and peripherallyadministered by bolus injection with from about 20 units to about 300units of a botulinum toxin, such a botulinum toxin type A, at or in thevicinity of the site of a prospective incision into the patient'sdermis. The botulinum toxin injection can be subcutaneous orintramuscular. The surgery is not carried out to treat or to alleviatepain which results from a hyperactive or hypertonic muscle because wehave surprisingly discovered that many types of pain which do not arisefrom or which do not result from a muscle spasm, can be significantlyalleviated by practice of our disclosed invention.

According to our invention, for relief from post-operative pain, apatient who is scheduled for surgery for the purpose of tumor removal,bone graft, bone replacement, exploratory surgery, wound closure, acosmetic surgery such as liposuction, or any of a myriad of other typesof possible (non-muscle disorder treatment) surgical procedures whichrequire one or more incisions into and/or through the patient's dermiscan be treated, according to our invention, by peripheral administrationof from about 0.01 U/kg to about 60 U/kg of a-botulinum toxin, such as abotulinum toxin type A or B. The duration of significant post-operativepain alleviation can be from about 2 to about 6 months, or longer.

A method within the scope of the present invention can provide improvedpatient function. “Improved patient function” can be defined as animprovement measured by factors such as a reduced pain, reduced timespent in bed, increased ambulation, healthier attitude, more variedlifestyle and/or healing permitted by normal muscle tone. Improvedpatient function is synonymous with an improved quality of life (QOL).QOL can be assesses using, for example, the known SF-12 or SF-36 healthsurvey scoring procedures. SF-36 assesses a patient's physical andmental health in the eight domains of physical functioning, rolelimitations due to physical problems, social functioning, bodily pain,general mental health, role limitations, due to emotional problems,vitality, and general health perceptions. Scores obtained can becompared to published values available for various general and patientpopulations.

EXAMPLES

The following non-limiting examples provide those of ordinary skill inthe art with specific preferred methods to treat non-spasm related painwithin the scope of the present invention and are not intended to limitthe scope of the invention. In the following examples various modes ofnon-systemic administration of a neurotoxin can be carried out. Forexample, by intramuscular bolus injection, by multiple subcutaneousinjections at dermal sites at and in the region of pain or byimplantation of a controlled release implant.

Example 1 Pain Alleviation by Peripheral Administration of BotulinumToxin Type A

Two experiments were carried out. Sprague-Dawley rats (about 300 toabout 350 grams) were used in both experiments. The neurotoxin used inboth experiments was BOTOX® (botulinum toxin type A purified neurotoxincomplex). In the first experiment there were 4 treatment (dose) groups:control (saline injected) rats (n=4), 7 U BOTOX®/kg rats (n=8), 15 UBOTOX® kg rats (n=5), and 30 U BOTOX®/kg rats (n=4). For the controlrats, 25 microliters of 0.9% saline solution was injected subcutaneouslyinto the plantar surface of the animal's hind paw. The site and route ofadministration of BOTOX® was the same as for the saline injectioncontrol group.

Five days after either the saline or BOTOX® injection, 50 microliters of5% formalin was injected at the site on each of the rats in all fourgroups where either saline or BOTOX® had been previously injected. Limblifting/licking by the subject animals was then recorded at 5 minuteintervals for one hour.

The second set of experiment involved the same protocol as did the firstexperiment. In the second experiment there were three treatment (dose)groups: control (saline-injected) rats (n=3), 3.5 U/kg rats (n=7), and 7U/kg rats (n=8); and the formalin test was conducted on the twelfth dayafter the original BOTOX® or saline injection.

The results of these two experiments are shown on FIGS. 1 and 2,respectively. The first 5 to 10 minutes can be referred to as phase 1,which is followed by phase 2. As shown by FIGS. 1 and 2, at both 5 daysand 12 days after injection, there was a significant dose dependant painalleviation in the BOTOX® treated animals.

Example 2 Peripheral Administration of a Botulinum Toxin to Alleviate aNon Spasm Pain

A 46 year old woman presents with pain localized at the deltoid regiondue to an arthritic condition. The muscle is not in spasm, nor does itexhibit a hypertonic condition. The patient is treated by a bolusinjection of between about 50 Units and 200 units of intramuscularbotulinum toxin type A. Within 1-7 days after neurotoxin administrationthe patient's pain is substantially alleviated. The duration ofsignificant pain alleviation is from about 2 to about 6 months. A painin the shoulder, arm, and hand due to osteoporosis, fixation of joints,coronary insufficiency, cervical osteoarthritis, localized shoulderdisease, or due to a prolonged period of bed rest can be similarlytreated.

Example 3 Peripheral Administration of a Neurotoxin to TreatPostherapeutic Neuralgia

Postherapeutic neuralgia is one of the most intractable of chronic painproblems. The patients suffering this excrucatingly painful processoften are elderly, has debilitating disease, and are not suitable formajor interventional procedures. The diagnosis is readily made by theappearance of the healed lesions of herpes and by the patient's history.The pain is intense and emotionally distressing. Postherapeuticneuralgia may occur any where, but is most often in the thorax.

A 76 year old man presents a postherapeutic type pain. The pain islocalized to the abdomen region. The patient is treated by a bolusinjection of between about The patient is treated by a bolus injectionof between about 50 Units and 200 units of botulinum toxin type Asubcutaneously to the abdominal region. Within 1-7 days after neurotoxinadministration the patient's pain is substantially alleviated. Theduration of significant pain alleviation is from about 2 to about 6months.

Example 4 Peripheral Administration of a Neurotoxin to TreatNasopharyngeal Tumor Pain

These tumors, most often squamous cell carcinomas, are usually in thefossa of Rosenmuller and may invade the base of the skull. Pain in theface is common. It is constant, dull-aching in nature.

A 35 year old man presents a nasopharyngeal tumor type pain. Pain isreported in the lower left cheek. The patient is treated by a bolusinjection of between about 10 units and about 35 units of botulinumtoxin type A intramuscularly to the cheek. Within 1-7 days afterneurotoxin administration the patient's pain is substantiallyalleviated. The duration-of significant pain alleviation is from about 2to about 6 months.

Example 5 Peripheral Administration of a Neurotoxin to Treat ChronicInflammatory Pain

A patient, age 45, presents with chronic inflammatory pain in the chestregion. The patient is treated by a bolus injection of between about 50Units and 200 units of intramuscular botulinum toxin type A. Within 1-7days after neurotoxin administration the patient's pain is substantiallyalleviated. The duration of significant pain alleviation is from about 2to about 6 months.

Example 6 Peripheral Administration of a Neurotoxin to Treat Pain Causedby Burns

A patient, age 51, experiencing pain subsequent to a severe andextensive first or second degree burns to the arm. The patient istreated by a bolus injection of between about 30 units to about 200units of botulinum toxin type A, subcutaneously to the arm. Within 1-7days after neurotoxin administration the patient's pain is substantiallyalleviated. The duration of significant pain alleviation is from about 2to about 6 months.

Example 7 Peripheral Administration of a Neurotoxin to Treat Joint Pain

A patient, age 63, suffering from joint pain resulting from arthritis.The patient is treated by a bolus injection of between about 30 Unitsand 150 units of intramuscular botulinum toxin type A into the region ofthe painful joint. Within 1-7 days after neurotoxin administration thepatient's pain is substantially alleviated. The duration of significantpain alleviation is from about 2 to about 6 months.

Example 8 Peripheral Administration of a Neurotoxin to TreatPost-Operative Pain

A patient, age 39, from 1 hour to ten days prior to surgery, is locallyand peripherally administered by bolus injection or subcutaneousinjection with from about 20 units to about 300 units of a botulinumtoxin, such a botulinum toxin type A, at or in the vicinity of the siteof a prospective incision into the patient's dermis. The botulinum toxininjection can be subcutaneous or intramuscular. The surgery is notcarried out to treat or to alleviate a muscle disorder, such as ahyperactive or hypertonic muscle. The duration of significantpost-operative pain alleviation is from about 2 to about 6 months.

Example 9 Treatment of Visceral Pain by Administration of a Neurotoxin

A male patient age 46 presents with chronic abdominal pain of visceralorigin but of unknown etiology. Tumor or duct constriction ishypothesized. Subcutaneous or intraorgan botulinum toxin, such as fromabout 20 units to about 300 units of a botulinum toxin-type A, isadministered subcutaneously or intraorgan (at the site of the perceivedpain). Within one to seven days the pain is substantially alleviated.The duration of significant pain alleviation is from about 2 to about 6months.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes peripheral administration methods to alleviatenon-muscle disorder related pain wherein two or more neurotoxins, suchas two or more botulinum toxins, are administered concurrently orconsecutively. For example, botulinum toxin type A can be administereduntil a loss of clinical response or neutralizing antibodies develop,followed by administration of botulinum toxin type E. Alternately, acombination of any two or more of the botulinum serotypes A-G can belocally administered to control the onset and duration of the desiredtherapeutic, result. Furthermore, non-neurotoxin compounds can beadministered prior to, concurrently with or subsequent to administrationof the neurotoxin to proved adjunct effect such as enhanced or a morerapid onset of denervation before the neurotoxin, such as a botulinumtoxin, begins to exert its therapeutic effect.

1. A method for treating a neuropathic pain that is a non-inflammatoryallodynia, the method comprising the step of peripheral administrationof a therapeutically effective amount of a botulinum toxin to a mammalwith the neuropathic pain that is a non-inflammatory allodynia, therebyalleviating the neuropathic pain treating the alledynia. 2-3. (canceled)4. The method of claim 1, wherein the botulinum toxin is selected fromthe group consisting of botulinum toxin types A, B, C₁, D, E, F and G.5. The method of claim 1, wherein the botulinum toxin is a botulinumtoxin type A. 6-8. (canceled)
 9. The method of claim 1, wherein theallodynia is alleviated for between about 1 month and about 6 months.10-11. (canceled)
 12. A method for alleviating a neurolathic pain thatis a non-inflammatory allodynia, the method comprising the step ofperipheral administration of a therapeutically effective amount of abotulinum toxin type A to a human patient with the neuropathic pain thatis a non-inflammatory allodynia, thereby alleviating the neuropathicpain. 13-27. (canceled)
 28. The method of claim 1, wherein theperipheral administration is by subcutaneous administration.
 29. Themethod of claim 1, wherein the peripheral administration is byintramuscular administration.
 30. The method of claim 12, wherein theperipheral administration is by subcutaneous administration.
 31. Themethod of claim 12, wherein the peripheral administration is byintramuscular administration.
 32. The method of claim 12, wherein theallodynia is alleviated for between about 1 month and about 6 months.33. A method for treating a neuropathic pain that is a non-inflammatoryallodynia, the method comprising the step of peripheral administrationof a therapeutically effective amount of a botulinum toxin type A to amammal with neuropathic pain that is a non-inflammatory allodynia,thereby alleviating the neuropathic pain for between about 1 month and 6months.
 34. The method of claim 33, wherein the peripheraladministration is by subcutaneous administration.
 35. The method ofclaim 33, wherein the peripheral administration is by intramuscularadministration.